June 15, 2013 (Vol. 33, No. 12)
Michael D. O’Neil
Tiny, subcellular membrane-bound vesicles called exosomes are periodically released by a wide variety of cell types, carrying along with them proteins, microRNA (miRNA), and mRNA fragments representative of their cells of origin.
It is thought that exosomes may shuttle information from cell to cell. For instance, it has been shown that exosomes can carry material from cancer cells that acts to suppress the immune system and stimulate angiogenesis, thus encouraging cancer growth.
Exosomes can be easily isolated from bodily fluids by ultracentrifugation and filtration, among other approaches, offering an attractive potential alternative to invasive biopsies for diagnostic and prognostic purposes.
While representative of the cell of origin, exosome cargo can over- or under-represent the molecular content of the originating cell. Consequently, it is believed that a selective packaging mechanism is involved in the loading of exosomes, though such a mechanism has not yet been demonstrated.
During the “International Society of Extracellular Vesicles” (ISEV) meeting held in Boston this April, GEN spoke with several scientists for whom sequencing the RNA content of exosomes is a key aspect of their work.
Novartis’ Maja M. Janas, Ph.D., is working to identify miRNAs commonly enriched or reduced in exosomes across eight different liver cancer cell lines as part of a larger effort to investigate the mechanism of selective miRNA sorting into exosomes.
Dr. Janas, a postdoctoral fellow at the Biologics Center, Novartis Institute for BioMedical Research, explained that she is looking for common RNA motifs, secondary structures, and proteins that bind these miRNAs. From there, she is working to identify miRNAs that are selectively enriched or reduced in exosomes from metastatic liver cancer cell lines, relative to those in nonmetastatic liver cancer cell lines.
To date, she has evaluated two liver cancer cell lines—one metastatic, one not. Dr. Janas said that deep sequencing of precursor and mature miRNA using the Illumina HiSeq platform revealed miR-21 was the most highly enriched miRNA in exosomes from the metastatic cell line, and that miR-378, miR-30a, miR-10a, and let-7i were found exclusively in the metastatic exosomes. Among those miRNAs observed exclusively in exosomes from the nonmetastatic cell line were miR-192, miR-191, miR-27b, and miR-101.
Dr. Janas intends to use these selectively exported miRNAs as reporters in an RNAi screening assay for factors regulating the exosomal miRNA pathway using a liver cancer donor and receptor cell-line system, she said. Interestingly, sequencing revealed more precursor miRNAs than mature miRNAs in exosome cargo, Dr. Janas reported, adding that she intends to investigate why.
Meanwhile, scientists at Life Technologies are profiling the RNA in exosomes released by HeLa cells.
In an talk discussing his team’s approach, Alexander “Sasha” Vlassov, Ph.D., senior staff scientists and manager, R&D, Life Technologies, noted that deep sequencing of RNA cargo, using either the Ion Torrent™ Personal Genome Machine or Ion Proton™ platform, has revealed that this content is primarily short RNA of 20–200 nucleotides in length, including miRNA, tRNA, and many short noncoding RNAs. The cargo also includes some full-length mRNA and ribosomal RNA, though little or no DNA.
Dr. Vlassov emphasized that exosomes are not the only entities containing extracellular circulating RNA. A significant fraction of circulating miRNA, for instance, is bound to proteins rather than encapsulated in exosomes, he said. On average, each exosome contains only 1 to 10 RNA molecules, he noted, assuming an average length of 100 nucleotides. However, taking into account that exosomes are present in very high numbers in body fluids (typically >109 per mL), as a population they are capable of inducing significant biological effects.
Speaking with GEN, Dr. Vlassov said the exosome field is “very exciting,” and that, given its potential for use in diagnostics and therapeutics for cancer and other diseases, Life Technologies is in the process of developing a complete arsenal of tools for exosome and microvesicle research.
Over at the University of Washington, Lucia Vojtech, Ph.D., and her colleagues are investigating why sexual transmission of HIV seems particularly resistant to vaccine-based prevention. Their hypothesis was that exosomes in human semen may carry a factor—or factors—that reduce the immune responsiveness of the genital mucosa.
It has previously been shown that factors in semen do directly influence early events in viral transmission and the immune response in the genital mucosa, but the exact components involved are not well understood.
Dr. Vojtech has found that exosomes are present in huge numbers in human semen, with individual ejaculates from six different donors containing, on average, 22 trillion exosomes. She also found that these exosomes rapidly enter antigen-presenting cells of the genital mucosa.
Further experiments showed that these exosomes could deliver functional miRNA to recipient cells. Deep sequencing using an Illumina HiSeq 2000 system revealed that the exosomes contained many different types of small RNAs, including miRNA, Y-RNA, piwi-RNA, and tRNA.
At least 162 miRNAs were present in all six samples, and), immunoregulatory activities have already reported or predicted for the most abundant miRNAs the researchers observed (including let-7b and miR-375). Dr. Vojtech noted that the miRNA content of the exosomes appeared highly selective, pointing to a selective packaging mechanism suggested by many in the field.
The University of Notre Dame’s Jeffrey Schorey, Ph.D., seeks to harness the power of exosomal RNA as a biomarker for Mycobacterium tuberculosis-infected macrophages. Speaking at the ISEV meeting, Dr. Schorey, professor and associate director, Institute for Global Health, Center for Rare and Neglected Diseases, emphasized the potential advantages of exosomes in tuberculosis (TB) diagnostics—namely, easy isolation from various bodily fluids, potential enrichment of TB components present in bodily fluids, and potential detection of TB RNA, TB proteins, and other TB components from a single source.
“For the sequencing of our cDNA library, which was made from the small RNA isolated from exosomes, we used the Roche 454 Genome Sequencer FLX+ instrument,” Dr. Schorey explained. “Our goal was to define not only the microRNAs present in the exosomes, but other small RNA species. One of the surprising findings from our sequencing results is that the exosomes contained M. tuberculosis RNA.”
From there, “we confirmed that the sequenced RNA was fragments of intact full-length transcripts as determined by RT-PCR of RNA isolated from exosomes released from infected macrophages,” he added.
To date, Dr. Schorey and his colleagues have shown that exosomes produced by M. tuberculosis-infected macrophages have a unique repertoire of miRNAs and mRNAs, and that these microvesicles carry mycobacterial proteins and mRNA transcripts and have immune modulatory activity.
Over at the VU University Medical Center in Amsterdam, immunology Michiel Pegtel, Ph.D. and his colleagues are examining how nonrandom small RNA identified in tumor exosomes show biomarker potential. They are taking a deep sequencing approach for these studies.
“In contrast to closed profiling technologies such as microarray and RT-PCR arrays, deep sequencing has literally opened our eyes on the complexity of the human transcriptome. Our previous work using individual quantitative RT-PCR could demonstrate that human B cells infected with the tumor virus Epstein Barr (EBV) secrete exosomes that contain fully mature and functional virus-encoded miRNAs,” Dr. Pegtel noted.
“At the ISEV meeting, Nobel laureate Dr. Phillip Sharp stressed the importance of quantitation in RNA research. I could not agree more as this applies directly to research concerning transfer of functional RNA via exosomes.”
Dr. Pegtel explained that in his team’s virus model, scientists could show that the transfer of relatively small copy numbers of miRNAs from an EBV-infected B cell into an uninfected recipient dendritic cell could directly influence gene regulation.
“Quantitation of RNA copy numbers in exosomes is important, but perhaps it is less important how many are in one or a thousand exosomes,” he said. “More important is what is actually transferred from one cell to another.”
He and his colleagues are now using the Illumina HiSeq 2000 and 100 paired-end protocol on both cancer cells and secreted exomes. Working in collaboration with the University of Granada’s Michael Hackenberg, Ph.D., assistant professor, Dr. Pegtel et al., have found that, aside from the presence of nearly all small RNA families, many smaller RNA fragments are preferentially sorted or excluded from secretion by exosomes indicating an underlying biology.
“Perhaps more interesting is that these small RNA fragments do not adhere to the general rules for Dicer-dependent miRNA processing,” Dr. Pegtel added. “Thus, deep sequencing has revolutionized not only what we know about RNAs inside cells, but also what is secreted into the extracellular milieu that can be used for diagnostics. Because of the quantitative nature of deep sequencing technology, we have gained additional insights into the potential mechanisms that control secretion of known and newly discovered RNAs via exosomes.”
He said that further research in this area will eventually tell scientists more about cell-cell communication in both health and disease. “Decoding the messages that are sent out by, for instance, cancerous cells will hopefully improve and simplify diagnosis such that we will all benefit from this exciting work,” Dr. Pegtel said.